Nephrolepis hirsutula 1

Chapter 3

Antibacterial and Phytochemical Screening of Ferns

52 Chapter 3

3.1 Introduction

Plants contain numerous biologically active compounds,

many of which have exhibited antimicrobial properties (Perumal

Samy and Ignacimuthu, 1998). Over the past few years, strains of

many pathogenic species resistant to all widely available antibiotics

have emerged and proliferated (Tomasz, 1994). The antibiotics are

sometimes associated with adverse effects on hosts, which include

hypersensitivity, immunosuppression, allergic reactions and

depletion of beneficial gut and mucosal microbes (Idose et al.,

1968). As in human, reports of antibiotic resistant bacteria

emerging in animal populations are appearing with increasing

frequency (Bates et al., 1993). In many parts of the world plants are

used for antibacterial, antifungal and antiviral medicine. The

extracts were used as a source of medicinal agents to cure urinary

tract infections, cervicitis, vaginitis and gastrointestinal disorders

(Caceres et al., 1990).

In this study few locally available ferns were screened for

their antibacterial activity; some of them had been described as

ethnobotanically relevant. 80% ethanol extracts of different plants

were investigated for antibacterial activity towards selected

bacterial strains.

3.2 Materials and Methods

3.2.1 Procurement of plant materials

Fresh specimens were collected based on their richness in

the month of November from the Vagamon Hills and nearby areas

of Kottayam District of Kerala State, India. Plants of limited

population were avoided in order to conserve them. Voucher

specimens identified by Dr. P.V. Madhusoodanan (Pteridologist and

Antibacterial and Phytochemical Screening of Ferns 53

Professor of Botany, Calicut University Kerala-673635, India.) were

deposited at the herbarium of Calicut University Herbarium (CALI).

Table 3.1 List of ferns selected for antibacterial screening and

their ethnobotanical importance.

N0

Botanical Name and Family (Voucher

Specimen No)

Medicinal uses of plants/their genus

1. Angiopteris evecta (G.

Forst.) Hoffm.

Angiopteridaceae

(TT 2049)

Aromatic oil from large sized fern used for

perfuming coconut oil in south sea Islands. In

Central India the rhizomes are used against

scabies (Vasudeva, 1999). Treatment of Cancer/

Sarcoma (Defilpps et al., 1998)

2. Osmunda regalis L.

Osmundaceae

(TT 2050)

Cultivated as ornamental and local tribals of

Madhya Pradesh, India utilised its first frond of

the year to cure toothache (Vasudeva, 1999).

3. Lygodium Flexuosum

(L.) Sw.

Schizaceae

(TT 2051)

Syn. Ophioglossum

fluxuosum L.

The plant has been described as an expectorant

and its roots extract in mustard oil is considered

an effective remedy for the treatment of wounds

and enzema. Leaf paste also applied over the skin

to treat skin disease (Nayar, 1959).

4.

Acrostichum aureum L.

Pteridaceae

(TT 2052)

Plant is used as styptic and anthelmintic. Also

used as a worm remedy and as an astringent in

haemorrhage (Chopra et al., 1956). Used in

traditional medicine of Nicobarese tribes (Dagar,

1989)

5. Pteris quadriaurita Retz.

Pteridaceae

(TT 2053)

Prteis aquilinia L. used as antihelmintic (Nayar,

1959). Decoction of fresh rhizome and fronds are

given in chronic disorders arising from

obstructions of viscera and spleen. (Chopra et al.,

1956).

54 Chapter 3

6. Cheilanthes bullosa

Kunze.

Sinopteridaceae

(TT 2054)

Syn. Cheilanthes

farinosa Kaulf var

flaccida Bedd.

Cheilanthes farinosa Kaulf The plant juice filtered

and mixed with rice starch is given thrice a day for

the discharge of yellowish urine in the doses of 20-

30 ml (Lal et al., 1996).

7. Cheilanthes tenuifolia

(Burm.f.) Sw.

Sinopteridaceae

(TT 2055)

Syn. Trichomanes

tenuifolia Burm.f.

Used by tribal as a general tonic (Nayar, 1959).

8. Doryopteris concolor

(Langsd. & Fischer) Kuhn

in Decken

Sinopteridaceae (TT 2056)

Syn. Pteris concolor

Langsd. & Fischer

Prteis aquilinia L. used as antihelmintic (Nayar,

1959). Decoction of fresh rhizome and fronds are

given in chronic disorders arising from

obstructions of viscera and spleen. (Chopra et al.,

1956).

9. Parahemionitis cordata

(Roxb. ex Hook. & Grev.)

Fraser-Jenk.

Hemionitidaceae (TT

2057)

Syn. Hemionitis cordata

Roxb. ex Hook. & Grev.

The Rabbits Ear Fern used in the treatment of

earaches and as a vermifuge. The whole plant

parts are used. (Vasudeva, 1999).

10. Pityrogramma austro-

americana Domin

Hemionitidaceae (TT

2058)

Syn. Pityrogramma

calomelanos var.

aureoflava (Hook.)

Weath. ex Baily

The Golden fern cultivated in the botanical

gardens as ornamental plants due to their

splendid beauty and grace (Vasudeva, 1999).


11. Pityrogramma

calomelanos (L.) Link

Hemionitidaceae (TT

2059)

Syn. Acrostichum

calomelanos L.

The decoction of rhizomes used as an anthelmintic

(Vasudeva, 1999). Smoke the leaf for colds in head

and chests (Caius, 1935).

12. Adiantum caudatum L.

Adiantaceae (TT 2060) Used in cough and fever (Vasudeva, 1999).

13. Adiantum latifolium

Lam.

Adiantaceae (TT 2061)

Syn. Adiantum

denticulatum Sw.

The whole plant parts are used as medicinal. Plant

parts are mucilaginous, pectoral, expectorant and

emmenagogue. The leaves are used as a febrifuge

and catarrhal affections. Used popularly as a

cough medicine and for all throat infections

(Nayar, 1959). In France large quantities are used

to prepare the famous “Sirop de capillare”.

14.

Adiantum lunulatum

Burm.f.


Syn. Adiantum

philippense L.

Rhizome used to cure glandular swelling

accompanied by fever. Juice of leaves used in

treatment of dysentery, diseases of blood, ulcers,

etc. In Ayurveda the plant is recommended to cure

for epilepsy. The spores are used in treatment of

leprosy and other skin diseases (Nayar, 1959).

Used as contraceptive agent to induce sterility

(Dhiman, 1998)

15. Adiantum raddianum C.

Presl.


Syn. Adiantum

cuneatum Langsd. &

Fisch.

Adiantum lunulatum rhizome used to cure

glandular swelling accompanied by fever. Juice of

leaves used in treatment of dysentery, diseases of

blood, ulcers, etc. In Ayurveda the plant is

recommended to cure for epilepsy. The spores are

used in treatment of leprosy and other skin

diseases (Nayar, 1959).

56 Chapter 3

16. Microlepia speluncae

(L.) T. Moore

Dennstaedtiaceae (TT

2064)

Syn. Polypodium

speluncae L.

Microlepia scaberula used against eye disease

(McClatchey, 1993) Used in traditional medicine of

Nicobarese tribes (Dagar, 1989)

17. Lindsaea ensifolia Sw.

Lindsaeaceae (TT

2065)

Syn. Schizoloma

ensifolium (Sw.) J.Sm.

in Hook.

Fronds of Sphenomeris chinensis, Lindsaeaceae are

used to produce red dye. The plants are used

internally for chronic enteritis (Fosberg, 1942).

18. Odontosoria chinensis

(L.) J.Sm.

Lindsaeaceae (TT 2066)

Syn. Trichomanes

chinensis L.

Used in the treatment of chronic enteritis (Nayar,

1959).

19. Nephrolepis hirsutula (G.

Forst.) C.Presl.

Oleandraceae (TT 2067)

Syn. Polypodium

hirsutulum G. Forst.

In Garhwal a decoction of fresh tubers and fronds

is given to cure cough (Shah and Singh, 1990).

Commonly cultivated in the botanical gardens as

ornamentals and as potted plants for indoor

decoration (Kholia and Punetha, 2005)

20. Dicranopteris linearis

var. linearis Holttum

Gleicheniaceae (TT

2068)

Syn. Polypodium

linearis Burm. f.

Young fronds with cow’s milk given in woman’s

sterility. The rhizomes are used as antihelmintic

(Vasudeva, 1999). Externally onto wounds

(Futscher, 1959) Used in traditional medicine of

Nicobarese tribes (Dagar, 1989)

21. Alsophila gigantea Wall.

ex Hook.

Cyatheaceae (TT 2069)

Syn Cyathea gigantea

(Wall. ex Hook.)

Holttum

Pith from the trunk is used as a food product

(Lawrence, 1951) Cyathea albo-setacea used in

traditional medicine of Nicobarese tribes (Dagar,

1989)


22. Alsophila spinulosa

(Wall. ex Hook.) R.M.

Tryon

Cyatheaceae (TT 2070)

Syn Cyathea spinulosa

Wall. ex Hook.

Ornamental plant (Vasudeva, 1999). Cyathea albo-

setacea used in traditional medicine of Nicobarese

tribes (Dagar, 1989)

23. Christella dentata

(Forssk.)Brownsey &

Jermy (TT 2071)

Thelypteridaceae

Syn. Polypodium

dentatum Forssk.

Used as cusion for cattle by Raji tribes (Pande and

Pangtey, 1987).

24. Christella parasitica (L.)

H. Lev.

Thelypteridaceae (TT

2072)

Syn. Polypodium

parasiticum L.

Used against wounds (Gogoi, 2002)

25. Cyclosorus interruptus

(Willd.) H.Ito


2073)

Syn. Pteris interrupta

Willd.

Pteris ensiformis used as astringent in Manipur

(Singh et al., 2001)

Boiled fronds of Cyclosorus used against boils,

ulcers and wounds (Powell, 1976).

26. Macrothelypteris

torresiana (Gaud.)

Ching


2074)

Syn. Polystichum

torresianum Gaud.

Cultivated as ornamentals in the botanical

gardens. As they generally spread by creeping

rhizomes (Vasudeva, 1999).

27. Asplenium formoum

Willd.

Aspleniaceae (TT 2075)

Genus Asplinium with antihelminthic, used

against Jaundice (Vasudeva, 1999).

58 Chapter 3

28. Diplazium esculentum

(Retz.) Sw.

Athyriaceae (TT 2076)

Syn. Hemionitis

esculenta Retz.

Young fronds are cooked and eaten by tribal

people for maintaining health (Kaushik and

Dhiman, 1995) Diplazium cochleata used against

epilepsy / Seizures (Dhiman, 1998)

29.

Dryopteris cochleata

(D.Don) C.Chr.

Dryopteridaceae (TT

2077)

Syn. Nephrodium

cochleatum D.Don.

A small portion of the rhizome of the plant is

powdered and taken with water twice a day in

rheumatism, epilepsy & leprosy (Shah and Singh,

1990). The whole plant is crushed in a bowl and

their extract is given twice a day orally, in case of

snake bite besides a paste of the plant is also applied

on the bite wound to prevent infection (Verma et al.,

1995). Rhizome used in swellings and pain and

antifungal properties (Asolkar et al., 1992).

30. Dryopteris hirtipes

(Blume) Kuntze

Dryopteridaceae (TT

2078)

Syn. Aspidium hirtipes

Blume

The juice obtained from the leaves is given in

epilepsy. And also used as an antibiotic.

(Vasudeva, 1999).

31. Tectaria coadunata

(Wall. ex Hook. & Grev.)

C.Chr.

Dryopteridaceae (TT

2079)

Syn. Aspidium

coadunatum Wall. ex

Hook. & Grev.

Tectaria wightii decoction of the rhizomes is given

to children, in stomach-ache in kumaon (Pande

et al., 1994).

32. Tectaria wightii (C.B.

Clarke) Ching

Dryopteridaceae (TT

2080)

Syn. Nephrodium

wightii C.B.Clarke

The decoction of the rhizomes is given to children,

in stomach-ache in Kumaon (Pande et al., 1994).

The plant used in asthma, bronchitis and in

getting relief from bites and stings by insects and

centipedes (Vyas and Sharma, 1988).Plants used

as antihelmintic (Kaushik and Dhiman, 1995)


33. Bolbitis x prolifera

(Bory) C.Chr.

Lomariopsidaceae (TT

2081)

Syn. Polybotrya

prolifera Bory in Bel.

General unspecified use of Moorea, French

Polynesia

Bolbitis lonchophora- Moorea (Nicole Baltrushes,

2006)

34. Blechnum orientale L.

Blechnaceae (TT 2082)

Syn. Blechnum orientale

var. grande B.K. Nair &

Geev.

The plants are used as poultice for boils by the

tribal people of central India. Rhizomes are used

as antihelminthic. Plants used as medicine for

diarrhoea and stomach disorders (Vasudeva,

1999).

35. Stenochlaena palustris

(Burm.f.)Bedd.

Syn. Polypodium

palustre Burm.f.

Blechnaceae (TT 2083)

Stenochlaena palustris are eaten as food (Powell,

1976). Stenochlaena palustris used in traditional

medicine of Nicobarese tribes (Dagar, 1989)

36. Drynaria quercifolia (L.)

J. Sm.

Polypodiaceae (TT

2084)

Syn. Polypodium

quercifolium L.

Rhizome used in treatment of typhoid, hectic fever

dyspepsia and cough and phthesis (Nayar, 1959).

Fronds used against swelling (May, 1978).

37. Microsorum punctatum

(L.) Copel.

Polypodiaceae (TT 2085)

Syn. Acrostichum

punctatum L.

Microsorum punctatum used in traditional

medicine of Nicobarese tribes (Dagar, 1989)

Microsorum grossum reported to be antiinflamatory

and antibacterial (Whistler, 1992)

38. Phymatosorus

nigrescens (Blume)

Pic.Serm.

Polypodiaceae (TT

2086)

Syn. Polypodium

nigrescens Blume

Used against nasal infections, the smoke of a

species of Polypodium (Powell, 1976).

60 Chapter 3

39. Pyrrosia heterophylla

(L.) M.G.Price.

Polypodiaceae (TT

2087)

Plants used in urinary calculus and rheumatism

(Caius, 1935).

40. Pyrrosia lanceolata (L.)

Farwell

Polypodiaceae (TT

2088)

Syn. Acrostichum

lanceolatum L.

Pyrrosia heterophylla used in urinary calculus and

rheumatism (Caius, 1935).

41. Pyrrosia porosa

(C.Presl) Hovenkamp

Polypodiaceae (TT

2089)

Syn. Niphobolus

porosus C.Pres

Pyrrosia heterophylla used in urinary calculus and

rheumatism (Caius, 1935).

42. Marsilea minuta L.

Masileaceae (TT 2090)

Syn. Marsilea crenulata

Desv.

Decoction of leaves mixed with ginger is given in

bronchitis and cough by the tribes (Vasudeva,

1999). Used to cure eye disease (Dhiman, 1998)

43. Salvinia molesta D.S.

Mitch.

Salviniaceae (TT 2091)

Weed (Mitchel, 1979)

44. Azolla pinnata R.Br.

Azollaceae (TT 2092)

Azolla filiculoides as a biofertiliser (Ripley et al.,

2003)

3.2.2 Preparation of plant extract

The air-dried plant material was ground and used for

preparing extracts. 20 grams of powdered plant material was

extracted with 80% ethanol by maceration and kept it for a

period of 24 hours at room temperature. The resultant extract

could be called as cold extract. The extracts were filtered using


Whatman No. I filter papers and each extract was concentrated

in a rotary evaporator to remove alcohol. Each cold extract was

dissolved in sterile distilled water to obtain a sample

concentration of 100 mg/ml (Cos et al., 2002).

3.2.3 Bacterial strains

The test organisms were collected from the culture collection

of the Institute of Microbial Technology (IMTECH) Chandigarh.

These include Micrococcus luteus (MTCC 6164), Escherichia coli

(MTCC 443), Staphylococcus aureus subsp. aureus (MTCC 96), and

Pseudomonas aeruginosa (MTCC 741) and Serratia marcescens

(MTCC 97) (referred to as MTCC strains henceforth). The bacteria

were sub-cultured on nutrient agar slants (Monica, 1985)

incubated at 37oC for 24 hours and stored at 4oC in the refrigerator

to maintain stock culture.

3.2.4 Maintenance of bacterial strains

Organisms were maintained by periodic subculture in

nutrient agar slants.

3.2.4.1 Nutrient agar slants

Medium and preparation

Beef extract 1 g

Yeast extract 2 g

Peptone 5 g

Sodium chloride 5 g

Agar 2 g

Water 1 L

62 Chapter 3

All the ingredients except agar were dissolved in distilled

water, gently heated, cooled and pH was adjusted to 7.4. Agar was

then added and dissolved by heating. The medium was then

dispensed in 5 ml quantities in test tubes. Sterilisation was done

by autoclaving at 121°C for 15 minutes (Monica, 1985). The

medium in the tubes was then allowed to solidify as slants.

3.2.5. The Inoculum

The nutrient broth cultures of the organisms, grown at 37°C

for approximately 3 to 4 hours were used as inocula.

3.2.6. Mueller-Hinton agar

Mueller-Hinton agar medium was employed for disc diffusion

sensitivity testing. Dehydrated medium supplied by Hi-Media

Laboratories Ltd., Bombay was used. The medium contained per

litre, infusion from 300 g beef, casein hydrolysate 17.5 g, starch

1.5 g, and agar 17 g (Monica, 1985). The medium was prepared by

dissolving the dehydrated mixture of ingredients in distilled water.

After boiling, pH was adjusted to 7.4 and sterilised by autoclaving

at 121°C for 15 minutes. The medium was poured in Petri plates.

3.2.7 In vitro antibacterial assay

The disc diffusion method as illustrated by Bauer et al.,

(1966) was used to determine the growth inhibition of bacteria by

plant extracts. Sterile liquid Mueller Hinton Agar media (pH 7.4 ±

2) was poured into sterile Petri dish, after solidification the

bacteria (1 ml bacterial broth of approximately105 cells) were

swabbed with a sterile needle under aseptic conditions.

Commercially available blank sterile discs (Hi Media Laboratories


Pvt. Ltd, Bombay) of 6 mm diameter were used in the study. Each

disc was impregnated with 20 µl containing about 1 mg of plant

extract. A disc with solvent blank (80% ethanol) was used as

control. The discs (including control) were used after drying them

in an incubator at 60°C to remove any trace of solvent. Discs were

introduced onto the surface of the medium. The plates were

incubated at 37oC for 24 hours to obtain inhibition zones.

Experiments were conducted in six replicates and average

inhibitory zone diameter was determined.

Antibacterial evaluation was conducted in two different

levels in the initial phase. Single ethanolic extract was analysed

for antibacterial activity against MTCC strains. In the second

phase, plants that showed greater level of antibacterial activity

were evaluated against twelve strains of pathogenic bacteria. The

extracts were prepared in different solvents of increasing polarity.

Plants that showed higher potential of antibacterial activity

in the first phase of screening were Osmunda regalis, Acrostichum

aureum, Lygodium flexuosum, Pteris quadri-aurita, Cheilanthes

bullosa, Cheilanthes tenuifolia, Adiantum latifolium, Lindsaea

ensifolia, Odontosoria chinensis, Dicranopteris linearis, Christella

dentata, Dryopteris cochleata, Blechnum orientale and Drynaria

quercifolia.

3.2.8 Plant extracts for second level of evaluation

The air-dried plant material was ground and used for

preparing extracts. About 50 g of powdered plant material was

successively extracted using petroleum ether (60-80oC) (relative

polarity 0.117), acetone (relative polarity 0.355), methanol

64 Chapter 3

(relative polarity 0.762) and water (relative polarity 1.000) for a

period of 24 hrs (Eloff, 1998). The extracts were filtered using

Whatman No. I filter papers and each extract was concentrated in

vacuum using rotary evaporator at 40oC to remove the last trace

of solvent. The solid material was dissolved in the same solvent to

obtain a sample concentration of 50 mg/ml.

3.2.9 Bacterial strains for the second level of evaluation

The test organisms were collected from the culture collection

of the Institute of Microbial Technology (IMTECH) Chandigarh,

India as described in section 3.2.3. The Sree Chitra Tirunal

Institute of Medical Sciences and Technology, Thiruvananthapuram

Kerala, India supplied some clinical isolates like Staphylococcus

albus, Salmonella typhi, Salmonella paratyphi, Citrobacter freundii;

Klebsiella pneumoniae, Shigella sonnei and Shigella dysentriae.

3.2.10 Antibacterial evaluation for the second level of evaluation

The disc diffusion method as illustrated by Bauer et al.,

(1966) as described in section 3.2.7 was used to determine the

growth inhibition of bacteria by plant extracts.

3.2.11 Preliminary detection of phytochemicals

Alkaloids were detected by Iodine Potassium iodide reagent

(Stahl, 1969), Dragendroff reagent (Thies and Reuther, 1954, Tyihak,

1964) and Margui reagent (Harborne, 1973). Presence of flavonoids

was detected by spraying with 25% aqueous solution of basic lead

acetate yielded fluorescence in long wave U.V light (Horhammer

et al., 1964). Spraying 1% aluminium chloride solution in ethanol

yielded yellow fluorescence in long wave U.V light (Gage et al., 1951)

indicated the presence of flavonoids. Phenols were detected by


spraying TLC plates with saturated aqueous silver nitrate solution;

light pink to deep green spots were yielded after spraying (Burke et

al., 1960). Undiluted Folin ciocalteu reagent was sprayed in order to

find out phenols (Keith et al., 1958). Stannic chloride was used to

detect titerpenes, sterols and steroids, phenol and poly phenols

(Scheideggeer and Cherbuliez, 1955). Anisaldehyde-sulphuric acid

reagent was used to detect terpenes, steroid and sugars (Stahl and

Kaltenbach, 1961).

3.3 Results and discussion

The activity was evaluated by measuring the inhibition zone

diameter around the disc and was recorded as follows: no obvious

growth inhibition (-); zone of inhibition with diameter 7 mm-10.99

mm (+); 11 mm-14.99 mm as ++ and zone of inhibition with

diameter 15-21 mm (+++). Inhibition zones were identified by

means of the disc diffusion method. The results are displayed in

Table 3.2. The results showed that some of the extracts like that of

Osmunda regalis and Lygodium flexuosum exhibited an

antibacterial effect towards all tested organisms. Therefore the

extracts were active against both gram-positive and gram-negative

bacteria. Plant extracts of Pteris quadri-auriata showed maximum

antibacterial activity i.e. greater than 20 mm. towards

Pseudomonas aeruginosa, a gram-negative bacterium (Table 3.2).

Pteris quadri-auriata showed moderate level of inhibition towards

Staphylococcus aureus, Escheichia coli and Micrococcus luteus. The

plant extract showed little sensitivity towards Serratia marcescens.

66 Chapter 3

Table 3.2 Antibacterial activity of ethanol extracts of ferns

The meaning of symbols used is as follows.

(-) = no obvious growth inhibition

(+) = zone of inhibition with diameter 7.00 -10.99 mm

[++] = zone of inhibition with diameter 11.00 -14.99 mm

(+++) = zone of inhibition with diameter 15.00-21.00 mm

Inhibition zone developed towards various bacterial strains

Family & Name of the plant

Part of the

plant used

MTC

C 9

6

MTC

C 4

43

MTC

C 7

41

MT

CC

616

4

MTC

C 9

7

Angiopteridaceae

Angiopteris evecta Fronds + + - - -

Osmundaceae

Osmunda regalis

Whole plant + + ++ ++ +

Schizaceae

Lygodium flexuosum

Whole plant + + ++ ++ +

Pteridaceae

Acrostichum aureum

Stem & Fronds ++ + ++ - -

Pteris quadri-aurita

Whole plant + + +++ + -


Sinopteridaceae

Cheilanthes bullosa

Whole plant - - ++ ++ +

Cheilanthes tenuifolia

Whole plant - ++ ++ - -

Doryopteris concolor

Whole plant - + + - -

Hemionitidaceae

Parahemionitis cordata

Whole plant - - + + -

Pitrogramma austroamericana

Whole plant + - + - +

Pitrogramma calomelanos

Whole plant + + + + -

Adiantaceae

Adiantum caudatum

Whole plant + - - + -

Adiantum latifolium

Whole plant + - ++ ++ -

Adiantum lunulatum

Whole plant + + + - -

Adiantum raddianum

Whole plant

+ - - - -

Dennstaedtiaceae

Microlepia speluncae

Whole plant + - - + -

68 Chapter 3

Lindsaeaceae

Lindsaea ensifolia

Whole plant + + +++ - +

Odontosoria chinensis

Stem & Fronds ++ ++ + + -

Oleandraceae

Nephrolepis hirsutula

Stem & Fronds + + - + ++

Gleicheniaceae

Dicranopteris linearis Rhizome ++ ++ + - +

Cyatheaceae

Alsophila gigantea Fronds + + - ++ -

Alsophila spinulosa Fronds + + - + -

Thelypteridaceae

Christella dentata

Whole plant

- - +++ ++ -

Christella parasitica Fronds - - + - -

Cyclosorus interruptus Fronds - - + - -

Macrothelypteris torresiana

Fronds + + + + -

Aspleniaceae

Asplenium formoum

Whole plant - + + + +


Athyriaceae

Diplazium esculentum

Whole plant - - + + -

Dryopteridaceae

Dryopteris cochleata Whole

plant ++ + ++ + -

Dryopteris hirtipes Fronds + - - - -

Tectaria coadunata

Whole plant - - + - -

Tectaria wightii

Stem & Fronds + - + + -

Lomariopsidaceae

Bolbitis prolifera

Stem &

Fronds - + + + -

Blechnaceae

Blechnum orientale Rhizome - ++ + ++ +

Stenochlaena palustris

Whole plant

- + + - +

Polypodiaceae

Drynaria quercifolia Rhizome +++ + ++ ++ +

Microsorum punctatum

Whole plant - + + + ++

Phymatosorus nigrescens

Whole plant - + + + +

Pyrrosia heterophylla


Pyrrosia lanceolata

Whole plant - - - - -

70 Chapter 3

Pyrrosia porosa


Masileaceae

Marsilea minuta


Salviniaceae

Salvinia molesta


Azollaceae

Azolla pinnata


Plants exhibited greater level of antibacterial activity were

Osmunda regalis, Lygodium flexuosum, Acrostichum aureum, Pteris

quadri-aurita, Cheilanthes bullosa, Cheilanthes tenuifolia, Adiantum

latifolium, Lindsaea ensifolia, Odontosoria chinensis, Dicranopteris

linearis, Christella dentata, Dryopteris cochleata, Blechnum orientale

and Drynaria quercifolia. Pseudomonas aeruginosa was found to be

the most sensitive organism to various extracts of ferns. Serratia

marcescens did not show any sensitivity to plant extracts.

The results of second level of screening of the plants for

antibacterial activities against various strains can be summarised

into following observations and assumptions.

Acetone and methanol extracts of most of the plants were more

active than petroleum ether and water extracts.

No antibacterial activity was recorded in the case of water

extracts of all plants. Water extraction was done at the end of


successive extractions and there was little possibility of finding

out active components in water extracts.

Petroleum ether extracts were less effective compared to

acetone and methanol extracts.

There is a general tendency that some medium-polar

compounds are involved in antibacterial activity.

Most of the extracts were active against Micrococcus luteus and

staphylococcus aureus.

Blechnum orientale L. showed broad range of antibacterial

activity against nine bacterial strains including gram-positive

and gram negative.

Maximum antibacterial activity was recorded for acetone

extracts derived from the rhizome extracts of Drynaria

quercifolia Dicranopteris linearis and Blechnum orientale.

The results of preliminary screening studies prompted us to

select Drynaria quercifolia for further studies.

Preliminary phytochemical tests demonstrated that the

occurrence of flavonoids, phenols and terpenes were more or less a

common feature of all plant extracts. However, plants differ

significantly in their activity against gram-positive and gram-

negative microorganisms. These differences may be attributed to the

fact that the cell wall in gram-positive bacteria consists of a single

72 Chapter 3

layer, whereas the gram-negative cell wall is a multi-layered

structure and quite complex (Madigan et al., 2000).

Experimental result of antibacterial activity of plant extracts

could be correlated with the ethnobotanical relevance of various

plants cited in Table 3.1. The presence of phenolics and flavonoids

was a general feature of different plant extracts (Table 3.3). While

alkaloid a more evolved compound generally observed in

Angiosperms was not observed as a common feature of Ferns.

Alkaloids are said to be more evolved in the sense that they are well

observed in highly evolved plant group, angiosperms. A few plants

like Microlepia speluncae, Nephrolepis hirsutula and Microsorum

punctatum showed the presence of alkaloids. Plants that showed

greater level of antibacterial activity exhibited the presence of both

flavonoid and terpenes. Both flavonoids and terpenes have

antibacterial properties (Vasange et al., 1997, Ramesh et al., 2001,

Shokeen et al., 2005). Since the medicinal plants studied appear to

have a broad antimicrobial activity spectrum, they could be useful in

antiseptic and disinfectant formulations as well as in chemotherapy

(Olukoya et al., 1993). Dicranopteris linearis is used traditionally

against wound infection (Futscher, 1959), the antibacterial potential

of the plant is confirmed by the present result especially towards

Staphylococcus aureus.

The antibacterial activity of Lindsaea ensifolia supported by

its ethnobotanical potential for chronic enteritis (Fosberg, 1942). The

optimal effectiveness of a medicinal plant may not be due to one

main active constituent, but to the combined action of different

compounds originallyin the plant (Bai, 1990). From this study we

conclude that promising plants exhibited broad spectrum of activity

against gram-positive and gram-negative bacteria and the results


confirm the use of these plants in traditional medicine for the

treatment of infections.

Table 3.3 Phytochemical contents of ethanolic extract of ferns

studied

The symbol [+] indicates present; and [-] indicates absence

Phytochemicals (+ present; - absent) Family and name of the

plant Alkaloids Flavonoids Terpenes Phenols

Angiopteridaceae

Angiopteris evecta - + + +

Osmundaceae

Osmunda regalis - + + +

Schizaceae

Lygodium flexuosum - + + +

Pteridaceae

Acrostichum aureum - + - +

Pteris quadri-aurita - + + +

Sinopteridaceae

Cheilanthes bullosa - - + +

Cheilanthes tenuifolia - + + +

Doryopteris concolor - + + +

74 Chapter 3

Hemionitidaceae

Parahemionitis cordata - + + +

Pitrogramma austroamericana

- + + +

Pitrogramma calomelanos - - - +

Adiantaceae

Adiantum lunulatum - + - +

Adiantum caudatum - + + +

Adiantum latifolium - + - +

Adiantum raddianum - + - +

Dennstaedtiaceae

Microlepia speluncae + + + +

Lindsaeaceae

Lindsaea ensifolia - + + +

Odontosoria chinensis - + + +

Oleandraceae

Nephrolepis hirsutula + + - +

Gleicheniaceae

Dicranopteris linearis - + + +


Cyatheaceae

Alsophila spinulosa - + + +

Alsophila gigantea - + + +

Thelypteridaceae

Christella dentata - + + +

Christella parasitica - + + +

Cyclosorus interruptus - + + +

Macrothelypteris torresiana - + + +

Aspleniaceae

Asplenium formoum - + + +

Athyriaceae

Diplazium esculentum - + + +

Dryopteridaceae

Dryopteris cochleata - + + +

Dryopteris hirtipes - + + +

Tectaria coadunata - + - +

Tectaria wightii - + - +

76 Chapter 3

Lomariopsidaceae

Bolbitis prolifera - + - +

Blechnaceae

Blechnum orientale - + + +

Stenochlaena palustris - + + +

Polypodiaceae

Drynaria quercifolia - + + +

Microsorum punctatum + + + +

Phymatosorus nigrescens - + + +

Pyrrosia heterophylla - - - +

Pyrrosia lanceolata - + + +

Masileaceae

Marsilea minuta - + + +

Salviniaceae

Salvinia molesta - - + +

Azollaceae

Azolla pinnata - - + +

77

Table 3.4 Detailed antibacterial activity of plants showed greater potential against pathogenic strains of bacteria.

The meaning of symbols used is as follows. (-) = no obvious growth inhibition; (+) = zone of inhibition with diameter 7.00-10.99 mm; (++) = 11.00-14.99 mm; (+++) = zone of inhibition with diameter 15.00-21.00 mm Abbreviations: PE - Petroleum ether Extract; AE - Acetone Extract; ME - Methanol Extract WE - Water Extract. S.al - Staphylococcus albus; S.au - Staphylococcus aureus; M.lu – Micrococcus luteus; S.ma - Serratia marcescens; S.ty - Salmonella typhi; S.pt - Salmonella paratyphi; C.fe - Citrobacter freundii; K.pn - Klebsiella pneumoniae; S.so - Shigella sonnei; S.dy - Shigella dysentriae; E. co - Escherichia coli; P.ae - Pseudomonas aeruginosa

Sl NO

Nam

e of

th

e pl

ant

and

Fam

ily

Part

u

sed

Ext

ra c

t u

sed

S.al

S.au

M.lu

S.m

a

S.ty

S.pt

C.f

r

K.p

n

S.so

S.dy

E.c

o

P.ae

PE - - - - - - - - - - - - AE + + - + - - - - - - - + ME - - - + - - + - - - - -

1. Osmunda regalis Osmundaceae

Whole plant

WA - - - - - - - - - - - - PE - - - - - - - - - - - - AE - ++ ++ + - - + - - - - - ME - - - - - - - - - - - -

2. Lygodium flexuosum Schizaceae

Whole plant

WA - - - - - - - - - - - - PE - - - - - - - - - - - - AE - + - - - - - - + - - - ME - ++ ++ - - - + - - - - -

3. Acrostichum aureum Pteridaceae

Stem & Fronds

WA - - - - - - - - - - - -


77

PE - - - - - - - - - - - - AE - - + + - - + - - - + + ME + + + - + - - - + - ++ -

4. Pteris quadri-aurita Pteridaceae

Whole plant

WA - - - - - - - - - - - - PE + - - - - - - - - - - - AE - + - + + - - + + - + + ME + - + - - - - - - - - -

5. Cheilanthes bullosa Sinopteridaceae

Whole plant

WA - - - + - - - - - - - - PE - - - - - - - - - - - - AE + - - - + - + - - - - - ME + - - + - - - + - - + -

6. Cheilanthes tenuifolia Sinopteridaceae

Whole plant

WA - - - - - - - - - - - - PE - - - - - - - - - - - - AE - + - - - + - + + - - + ME + - + - - - - - - - - ++

7. Adiantum latifolium Adiantaceae

Whole plant

WA - - - - - - - - - - - - PE - - - - - - - - - - - - AE - ++ - - - - - - - - - - ME + - ++ - - - - - - - - +

8. Lindsaea ensifolia Lindsaeaceae

Whole plant

WA - - - - - - - - - - - - PE - - - - - - - - - - - - AE + - ++ - - - - - - - + - ME - + - + - + - + - - + -

9. Odontosoria chinensis Lindsaeaceae

Stem & Fronds

WA - - - - - - - - - - - -

78

Chapter 3

79

PE - - - - - - - - - - - - AE - ++ + + - - ++ - - + - + ME + - - - - - - - + - + + 10.

Dicranopteris linearis Gleicheniaceae

Rhizome

WA - - - - - - - - - - - -

PE - - - - - - - - - - - AE + - + - - - + - - - + - ME - + - - - - - - + - + -

11.

Christella dentata Thelypteridaceae

Whole Plant

WA - - - - - - - - - - - - PE - - - - - - - - - - - - AE + - + - - - - - - - - - ME - + - - - - - - - - - -

12. Dryopteris cochleata Dryopteridaceae

Stem Fronds

WA - - - - - - - - - - - - PE - - - - - - - - - - - - AE - + + - + - ++ - - - + ++ ME + - + - - - - + - + + -

13. Blechnum orientale Blechnaceae

Rhizome

WA - - - - - - - - - - - - PE - - + - - + ++ - - + - - AE - ++ + + - + ++ - - ++ - ++ ME - + - - + - + - - + - +

14.

Drynaria quercifolia Polypodiaceae

Rhizome

WA - - - - - - - - - - - -


79

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